The versatility and resourcefulness of microbes in developing resistance to various therapies are widely recognized. Although chemical modifications of existing drugs and the development of novel inhibitors against a handful of previously established targets has proven to be successful in the short term, it is also apparent that new drug targets need to be explored to maintain and extend efficacious antibacterial therapy in the long run [1]. The need for new targets is further exacerbated by the emergence of bacterial pathogens with natural resistance to existing antibiotics and by a potential threat of pathogens with engineered antibiotic resistance.
NAD(P) biosynthesis as a promising, albeit relatively unexplored target pathway for the development of novel antimicrobial agents [2-4]. Cofactors of the NAD pool are indispensable as they are involved in hundreds of redox reactions in the cell. Additionally, NAD is utilized as a cosubstrate by a number of non-redox enzymes (e.g., by bacterial DNA ligases and protein deacetylases of the CobB/Sir2 family). This dictates the need to maintain NAD homeostasis via its active resynthesis and recycling of NAD degradation products. Recently, a number of insightful reviews have emphasized the potential of NAD(P) biosynthetic enzymes as drug targets for the treatment of cancer, autoimmune diseases, and neurodegenerative disorders [5-8]. Although the early steps in NAD biogenesis and recycling vary substantially between species, the enzymes driving the downstream conversion of nicotinic acid mononucleotide (NaMN) to NAD and NADP are present in nearly all analyzed bacterial genomes[2, 9]. Therefore, all three enzymes of this pathway—NaMN adenylyltransferase (EC 2.7.7.18), NAD synthetase (EC 6.3.1.5) and NAD kinase (EC 2.7.1.23) (encoded by the conserved genes nadD, nadE and nadF, respectively), represent promising broad-spectrum antibacterial targets. The observed essentiality of the respective genes is due to bacteria being unable to uptake phosphorylated pyridine nucleotides [2, 3]. Recent progress in the development of inhibitors targeting the last two enzymes, NadE [10-12] and NadF [13, 14], provides additional validation of NAD biosynthesis as a target pathway.
NadD converts NaMN, the first intermediate shared by the most common de novo and salvage/recycling routes, to nicotinic acid adenine dinucleotide (NaAD). Therefore, this enzyme should be indispensable in all bacterial species that utilize one or both of these routes for NAD biosynthesis. This is consistent with gene essentiality data for a number of bacterial species (as reviewed in [3, 16]). For example, the nadD gene was shown to be essential for survival in Staphylococcus aureus and Streptococcus pneumoniae that are fully dependent on niacin salvage (via PncA-PncB route). It is also essential in Escherichia coli and Mycobacterium tuberculosis, organisms that harbor both the de novo (NadB-NadA-NadC) and the salvage pathways. Remarkably, it has been recently demonstrated that NAD downstream pathway holds as an attractive target in both actively growing and nonreplicating pathogens [17]. NadD is present in nearly all important pathogens with only a few exceptional cases, such as Haemophilus influenzae which lacks most of NAD biosynthetic machinery and is dependent on salvage of the so-called V-factors [18].
Many representatives of the NadD family from pathogenic and model bacteria have been characterized mechanistically and structurally [19-24]. All of these enzymes have a strong substrate preference for NaMN over its amidated analog, NMN. On the other hand, all three isoforms of the functionally equivalent human enzyme (hsNMNAT-1, hsNMNAT-2 and hsNMNAT-3) have an almost equal catalytic efficiency for either substrate, NaMN or NMN [25, 26]. The observed difference in substrate specificity reflects the dual physiological role of the human enzyme (hereafter referred to as hsNMNAT) in the adenylation of both intermediates contributing to NAD biogenesis [7, 27]. Notably, among the three bacterial enzymes of the target pathway, NadD has the lowest sequence similarity to its human counterparts [3]. Comparative analysis of 3D structures of bacterial NadD and hsNMNAT revealed significant differences between their active site conformations [15], which are likely responsible for their distinct substrate specificities, thus opening an opportunity for selective targeting.
It is apparent that there is a need in the art for novel antimicrobial agents. To this end, the inventors have selected the NadD enzyme as a target for the development of specific inhibitors based on a number of criteria such as essentiality, broad conservation and structure-function distinction from its human counterpart.